The in vitro metabolism of cyclophosphamide will be studied with regard to stereochemical aspects of its conversion to aldophosphamide by hepatic microsomal mixed-function oxidase. Stereospecificity and the absolute stereochemistry of this primary cytotoxic activation process will be explored by (1) determining the ratio of aldophosphamide enantiomers which are produced by metabolism of racemic cyclophosphamide, and (2) by kinetic comparison of the enzymatic catalysis parameters (Km, Vmax) for enantiomerically pure samples of cyclophosphamide. Possible stereospecific enzyme inhibition of this cyclophosphamide metabolic process is subject to test by measurement of Ki values using known mixtures of (R)- and (S)-cyclophosphamide. To accomplish these main objectives, a number of stereospecific synthetic routes for isolation of enantiomericaly pure cyclophosphamide and aldophosphamide are proposed, together with a radio-chromatographic analytical technique for direct assay of aldophosphamide enantiomeric excess.